Although our current knowledge about mammalian DNA replication suggests that many enzymes and other proteins are involved, none have until recently been shown to be required. To determine which enzymes are required for DNA replication, I propose to analyze the replication in vitro of Simian virus 40 (SV40) chromosomes. Replicating SV40 chromosomes can be isolated in a form which allows DNA synthesis to continue. The relative simplicity of this system should enable us to fractionate the enzyme bound to these chromosomes and determine their role(s) in DNA replication. The chromosomes will be assayed for the presence of enzymes believed to be involved in replication. For each enzyme detected, we will determine whether it is preferentially associated with replicating SV40 chromosomes. We will also examine whether the removal or inhibition of each enzyme results in an alteration in the process of DNA replication. If an alteration is detected it provides excellent evidence for the role(s) of that enzyme in the replication process; if no effect is found, the possibility of adventitious association will be tested.